Project Summary/Abstract The brain is a highly inter-connected network system, with distributed brain regions orchestrating to maintain normal brain function and mediate complex behavior. It is becoming increasingly clear that altered brain network properties underlie mental illness, but the neural substrates causing these large-scale network changes remain unknown. A key hypothesis is the dysfunction of brain hub regions. Hubs are brain regions that have high degrees of connections with the rest of the brain network. Because of their central roles, dysfunction of hub nodes can change global integrative process, and has been hypothesized to be a direct cause of altered brain network function and pathophysiology of brain disorders. However, directly testing this hypothesis in humans is challenging, as selectively manipulating activity in a hub and dissecting its causal impact on brain networks is difficult. We will bridge this critical gap using cutting-edge tools to manipulate the activity of a hub, and monitor the impact of these manipulations on brain networks using resting state functional magnetic resonance imaging (rsfMRI) and behavior in an awake rat model. In the current grant cycle we have established the rsfMRI approach in awake rats, which allows us to reliably measure resting-state functional connectivity (RSFC) and characterize brain network properties in rats. We have built on our awake rat rsfMRI approach by incorporating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), optogenetics, electrophysiology and animal behaviors. With these capacities, we can causally manipulate the activity in a hub brain region, and measure the corresponding brain-wide network reconfigurations. Using optogenetics, we can manipulate neural activity on the millisecond timescale, and using DREADDs, we can manipulate neural activity on the times scales of hours to days. Finally, concurrent electrophysiology-fMRI will allow us to directly relate brain network and behavioral changes to their neural basis. Our goal is to elucidate the causal impact of manipulating hub region activity on brain network organization, function and behavior in awake rats using rsfMRI, DREADDs, optogenetics and electrophysiology. In Aim 1, we will document changes in network properties including the network topological organization and brain-wide RSFC dynamics induced by semi-acute suppression of a brain hub. With the cell- type specificity of DREADDs, we will examine the role of the balance of excitation and inhibition in a hub in brain network dynamics. To elucidate the relationship between activity of a network node and specific brain network function, in Aim 2 we will dissect the functional role of each node in the default mode network and related behaviors in rats. In Aim 3, we will further determine the impact of chronic suppression of a hub on long-term network reorganization and behavior. Successful completion of the proposed research will elucidate the causal relationship between short-term and long-term dysfunction of a hub and brain network reconfigurations, which will help understand the neural substrates causing large-scale brain network changes.